Cardiovascular components for transplantation and methods of making thereof

ABSTRACT

Cardiovascular components such as biocompatible heart valves and annular sewing rings are disclosed, as well as, methods for making the same. The heart valves include biodegradable polymer fiber scaffolds and collagen. Also disclosed are donor aortic heart valves processed without the use of crosslinking chemicals.

BACKGROUND OF THE INVENTION

[0001] The heart includes four natural valves that function to regulateflow direction as blood is pumped between the lungs and the variousblood vessels. The mitral and tricuspid valves, which are known as theatrioventricular or intraflow valves operate to prevent backflow intothe atria during ventricular contraction while permitting blood to flowtherethrough during ventricular relaxation. The aortic and pulmonaryvalves are known as semilunar or outflow valves and are located whereblood leaves the heart.

[0002] Semilunar valves consist of three membranous cup-like structuresor cusps attached, at the same level, to the wall of a cylindricalaortic vessel so that the cusps press on each other when they are filledwith blood, preventing backflow in diastole. The direction of blood flowis upward. On contraction of the vessel, that is during systole, thecusps are pressed against the vessel wall by the force of blood flowingpast the attached edges of the cusps toward the free edges of the cusps,allowing the blood to flow freely.

[0003] Each open pocket of the semilunar valve defines a volume calledthe aortic sinus which is filled with blood when the valve is closed. Ifthe leaflet is cut away from the wall of the aorta it can be spread outin the form of a flat hemicircular membrane. The hemicircle is the edgeof the leaflet which is attached to the wall of the aorta while the topmore or less linear edge was the free edge of the leaflet. Each end ofthe leaflets called a commisure. The work of A. A. H. J. Sauren (TheMechanical behavior of the Aortic Valve (PhD thesis) Eindhoven, TheNetherlands: Eindhoven Technical University, 1981), which isincorporated herein by reference, has shown in whole mounts of leafletsthat the supporting scaffold of the leaflet consists of collagen fibers,having fractile properties, which extend from one commisure to the otherproviding support for the applied load of blood. Equations whichdescribe the fiber system of the leaflet have been derived by C. S.Perkin and D. M. McQueen (Mechanical equilibrium determines the fractilefiber architecture of aortic heart valve leaflets. Am. J. Physiol. 266,H319-H328, 1994) from their function which is to support a uniform loadwhen the aortic valve is closed. What they find is a single parameterfamily of collagen fibers with fractile properties which compare closelywith the whole mount fiber preparations. Their work serves as the basisfor creating a digital program which a textile machine, or a sewingmachine, could use to reproduce an approximation of the fiber scaffoldof the valve leaflet.

[0004] Histologically the leaflet consists of three tissue layers, thefibrosa, the spongiosa and the ventricularis. The fibrosa of the leafletfaces the aortic wall, enclosing the fiber system described above; thefiber scaffold is arranged in corrugated fashion permitting radialexpansion of the valve leaflet. Adjacent to the fibrosa is thespongiosa, a loosely organized connective tissue with collagen elastin,proteoglycans and mucopolysaccharides. Furthest away from the aorticwall is the ventricularis consisting of a sheet of elastin thought toprovide the tensile recoil needed to maintain the corrugated shape ofthe fibrosa. The surfaces of the leaflets in contact with the blood arecovered by a layer of endothelial cells.

[0005] Heart valves, e.g., semilunar valves, are deformed by a varietyof pathological processes. In many cases the diseased or defective valvecan be surgically removed and replaced with a prosthetic valve. Two maintypes of artificial valves exist: (1) mechanical valves made from metalor plastic material; and (2) valves made from animal tissue.

[0006] Artificial valves, whether mechanical or made from animal tissue,have serious drawbacks. For example, mechanical valves carry asignificant risk of thrombus formation. Also, the stress associated withthe junction between the stent or frame and the biological portion ofthe bioprosthetic valve appears to be involved in structural failureover time. Valves made from animal tissue are typically crosslinked withchemicals, e.g., glutaraldehyde during processing. Treatment of theanimal tissue with glutaraldehyde causes calcification and/or thestructural breakdown of the tissue, thus, reducing the area available asbinding sites for human host cells. In addition, both mechanical valvesand valves constructed from animal tissue do not have the capacity togrow, i.e., these types of valves can neither be occupied or remodeledby host cells nor can they be biologically integrated.

[0007] A need exists, therefore, for an improved prosthetic heart valvethat overcomes or minimizes the above-mentioned problems.

SUMMARY OF THE INVENTION

[0008] The invention features novel biocompatible cardiovascularcomponents, e.g., semilunar heart valves, for transplantation. Theinvention also features methods for constructing these novelbiocompatible cardiovascular components which preserve the nativity ofthe biological materials used. In addition, the invention features anovel annular sewing ring for attachment of a cardiovascular componentto the aortic wall of a host. The components can be used in vitro, forexample, for model systems for research, or in vivo as prostheses orimplants to replace diseased or defective heart valves. In either case,the valves can be seeded with cells, e.g., spongiosa cells, fibrosacells, ventricularis cells, smooth muscle cells, and/or endothelial andmesothelial cells.

[0009] In one aspect of the invention, the cardiovascular component is asemilunar valve which includes a biodegradable polymer fiber scaffold,e.g., a biopolymer fiber scaffold, and collagen. In a preferredembodiment, the collagen is fetal porcine collagen. In another preferredembodiment, the collagen is fibrillar collagen. In yet another preferredembodiment, the biopolymer fiber scaffold is a collagen biopolymerscaffold.

[0010] In another aspect of the invention, the cardiovascular componentis a semilunar valve which includes a biodegradable polymer fiberscaffold, e.g., a biopolymer fiber scaffold, and collagen wherein thebiopolymer scaffold fiber is derived from an aortic porcine valveprocessed in the absence of a crosslinking agent, e.g., glutaraldehyde.

[0011] In yet another aspect of the invention, the cardiovascularcomponent is a semilunar valve which includes a biodegradable fiberscaffold, e.g., a biopolymer fiber scaffold, and collagen wherein thescaffold has a structure determined by a digital program.

[0012] The invention further pertains to a method of making a semilunarheart valve, comprising the steps of: (a) assembling a mold whichreplicates the structure of a semilunar heart valve having between twolateral edges a hollow representing the aortic root and hollowsrepresenting a plurality of leaflets with outer and inner surfaces, theinner surfaces connecting with the hollow representing the aortic root,thus, forming the intimal surface of the aortic root; (b) covering theintimal surface of the hollow representing the aortic root, i.e., thesurface of the hollow representing the aortic root which connects, withthe hollow representing the valve leaflets, and the outside surface ofthe hollow representing the valve leaflets, i.e., the surface away fromthe aortic wall with a biodegradable polymer fiber scaffold; (c) fillingthe hollow representing the aortic root and the hollows representing theplurality of leaflets with collagen, e.g., fetal porcine collagen,fibrillar collagen e.g., liquid dense fibrillar collagen; and (d)freeze-drying the polymer fiber scaffold and the liquid dense collagenforming a tissue with two lateral edges.

[0013] The invention still further pertains to an annular sewing ringfor attachment of a heart valve to the aortic wall of a host whichincludes a biopolymer cloth and a biopolymer rope shaped in a circle,wherein the biopolymer cloth is wrapped around and stitched to thebiopolymer rope.

[0014] The invention yet further pertains to a semilunar heart valvemade according to a method which includes the steps of: (a) assembling amold which replicates the structure of a semilunar heart valve havingbetween two lateral edges a hollow representing the aortic root andhollows representing a plurality of leaflets with outer and innersurfaces, the inner surfaces connecting with the hollow representing theaortic root; (b) covering the intimal surface of the hollow representingthe aortic root and the outside surface of the hollow representing theplurality of leaflets with a biodegradable polymer fiber scaffold; (c)filling the hollow representing the aortic root and the hollowsrepresenting the plurality of leaflets with liquid dense collagen; and(d) freeze-drying the polymer fiber scaffold and the liquid densecollagen forming a tissue with two lateral edges.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the aorta slit open longitudinally and laid flat sothat the structure of the valve leaflets of the semilunar valve can bedisplayed.

[0016]FIG. 2 shows the valve leaflets of the semilunar valve from belowin the closed condition; filled with blood and pressing on each otherthereby preventing backflow.

[0017]FIG. 3 shows the valve leaflets of the semilunar valveschematically in longitudinal section.

[0018]FIG. 4 shows the mold design for constructing a semilunar heartvalve.

[0019]FIG. 4A shows the back cover of the mold which represents theoutside of the aorta.

[0020]FIG. 4B shows the front cover of the mold which represents theinside of the aorta displaying the attached leaflet molds.

[0021]FIG. 4C shows the back side of the front cover of the molddisplaying the back side of the leaflet molds as they join with theaorta.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

[0023] The present invention features novel biocompatible cardiovascularcomponents for transplantation, e.g., heart valves, e.g., semilunarheart valves. The term “biocompatible” as that term is used herein,means exhibition of essentially no cytotoxicity while in contact withbody fluids or tissues. “Biocompatibility” also includes essentiallyonly minimal interactions, i.e., interactions leading to immunerejection or to persistent inflammation responses, with recognitionproteins, e.g., naturally occurring antibodies, cell proteins, cells,and other components of biological systems. The invention also featuresmethods of making these components which preserves the nativity of thebiological material comprising the components.

[0024] In one aspect of the invention, the cardiovascular component is aheart valve, e.g., a semilunar heart valve which includes abiodegradable polymer fiber scaffold, e.g., a biopolymer fiber scaffold,and collagen. A semilunar heart valve is composed of three membranouscup-like structures or cusps attached, at the same level, to the wall ofa cylindrical arterial vessel, e.g., the aorta, so that the cusps presson each other when they are filled with blood, preventing backflow indiastole. Methods for making the polymer fibers which comprise thepolymer fiber scaffolds are taught in U.S. Pat. No. 5,851,290, entitled“Apparatus and Method for Spinning and Processing Collagen Fiber,” whichis incorporated herein by reference.

[0025] The term “biodegradable polymers,” as that term is used herein,includes any polymer that naturally degrades or breaks down over time byhydrolysis, for example, poly-α-hydroxyesters such as poly-1-lactic acidand poly-1-glycolic acid, polydioxinone, polyvinyl alcohol, surgicalgut, and combinations thereof, or which degrades over time by enzymaticaction, for example, biopolymer, e.g., collagen.

[0026] A biopolymer is a naturally occurring polymeric substance formedfrom individual molecules in a biological system or organism.Biopolymers can also be man-made by manipulation of the individualmolecules once obtained outside the biological system or organism. Thebiopolymer is suitable for introduction into a living organism, e.g., amammal, e.g., a human. The biopolymer is non-toxic and bioabsorbablewhen introduced into a living organism and any degradation products ofthe biopolymer should also be non-toxic to the organism. The biopolymersof the invention can be formed into cardiovascular components, e.g.,heart valves, e.g., semilunar heart valves, which include biocompatiblefibers, e.g., collagen fibers, biocompatible fabrics, e.g., collagenfabrics. Examples of molecules which can form biopolymers and which canbe used in the present invention include collagen, laminin, elastin,fibronectin, fibrinogen, thrombospondin, gelatin, polysaccharides,poly-1-amino acids and combinations thereof. In one embodiment, acombination or mixture of one or more biopolymers can be used to formthe cardiovascular components, e.g., heart valves, e.g., semilunar heartvalves, of the invention. For example, a combination of laminin and typeIV collagen can be used to form the biopolymer fibers described herein.A preferred molecule for biopolymer production is collagen.

[0027] Preferred sources of molecules which form biopolymers includemammals such as pigs, e.g., near-term fetal pigs, sheep, fetal sheep,cows, and fetal cows. Other sources of the molecules which can formbiopolymers include both land and marine vertebrates and invertebrates.In one embodiment, the collagen can be obtained from skins of near-term,domestic porcine fetuses which are harvested intact, enclosed in theiramniotic membranes. Collagen or combinations of collagen types can beused in the cardiovascular components, e.g., heart valves, e.g.,semilunar heart valves described herein. A preferred type of collagen isporcine fetal collagen. Another preferred type of collagen is fibrillarcollagen, e.g., fibrillar collagen can be produced by processing asolution of monomeric liquid collagen, e.g., non-polymeric liquidcollagen. Fibrillar collagen is a type of collagen which containsfibrils. The language “fibrillar collagen” or “collagen microfibril” isart recognized and is intended to include collagen in the form describedin Williams, B. R. et al. (1978) J. Biol. Chem. 253 (18):6578-6585 andU.S. patent application Ser. No. 08/910,853, filed Aug. 13, 1997,entitled “Compositions, Devices, and Methods for Coagulating Blood” byEugene Bell and Tracy M. Sioussat, the contents of which areincorporated herein by reference. In a preferred embodiment, thecollagen microfibrils are prepared according to the methods taught inU.S. patent appln. 60/095,627, entitled “Bone Precursor Compositions,”which are incorporated herein by reference. Liquid dense fibrillarcollagen is fibrillar collagen in a liquid form which can be dried to adense fibrillar tissue, e.g, a matt. Biopolymer and collagen matts aredescribed in copending patent application Ser. No. 09/042,549, entitled“Biopolymer Matt for Use in Tissue Repair and Reconstruction,” thecontents of which are incorporated herein by reference.

[0028] Examples of collagen or combinations of collagen types includecollagen type I, collagen type II, collagen type III, collagen type IV,collagen type V, collagen type VI, collagen type VII, collagen typeVIII, collagen type IX, collagen type X, collagen type XI, collagen typeXII, collagen type XIII, collagen type XIV, and collagen type XVII. Apreferred combination of collagen types includes collagen type I,collagen type III, and collagen type IV.

[0029] Preferred mammalian tissues from which to extract the moleculeswhich can form biopolymer include entire mammalian fetuses, e.g.,porcine fetuses, dermis, tendon, muscle and connective tissue. As asource of collagen, fetal tissues are advantageous because the collagenin the fetal tissues is not as heavily crosslinked as in adult tissues.Thus, when the collagen is extracted using acid extraction, a greaterpercentage of intact collagen molecules is obtained from fetal tissuesin comparison to adult tissues. Fetal tissues also include variousmolecular factors which are present in normal tissue at different stagesof animal development.

[0030] In a preferred embodiment, the cardiovascular components, e.g.,heart valves, e.g., semilunar heart valves, are collagen cardiovasularcomponents, e.g., collagen heart valves, e.g., collagen semilunar heartvalves. Collagen solutions can be produced by salt extraction, acidextraction, and/or pepsin extraction from the starting material. In apreferred embodiment, the collagen used is produced by sequentiallypurifying two forms of collagen from the same collagen-containingstarting material. First, intact collagen is acid extracted from thestarting material, the extract is collected and collagen is prepared asa collagen solution, e.g., by precipitating the collagen with sodiumchloride and solubilizing the collagen in a medium having an acidic pH.Meanwhile, truncated collagen, i.e., collagen from which theteleopeptides have been cleaved or partly cleaved leaving only thehelical portion or the helical portion with some telopeptides, isextracted from the starting material using enzyme, e.g., an enzyme whichis functional at an acidic pH, e.g., pepsin, extraction. Then, thecollagen from this pepsin extract is purified separately by similarmethods as from the first extract.

[0031] Proteins necessary for cell growth, morphogenesis,differentiation, and tissue building can also be added to the biopolymermolecules or to the biopolymer fibrils to further promote cell ingrowthand tissue development and organization within the cardiovascularcomponents, e.g., hearts valves, e.g., semilunar heart valves. Thephrase “proteins necessary for cell growth, morphogenesis,differentiation, and tissue building” refers to those molecules, e.g.,proteins which participate in the development of tissue. Such moleculescontain biological, physiological, positional, and structuralinformation for development or regeneration of the tissue structure andfunction. Examples of these macromolecules include, but are not limitedto, sonic hedgehog; NK-2, XNKx-3.3 (tinman), hCsx and Gax homeobox geneproducts; TGFbeta, VEGF, FGF, IGF, PDGF, BMP4 cytokine proteins, growthfactors, extracellular matrix proteins, proteoglycans,glycosaminoglycans and polysaccharides. Alternatively, thecardiovascular components, e.g., heart valves, e.g., semilunar heartvalves of the invention can include extracellular matrix macromoleculesin particulate form or extracellular matrix molecules deposited by cellsor viable cells or deliberately added to the valve scaffold. Methods forprocessing tissues for making extracellular matrix macromolecules inparticulate form are taught in U.S. Pat. No. 5,800,537, entitled “AMethod and Construct for Producing Graft Tissue From ExtracellularMatrix,” the contents of which are incorporated herein by reference.

[0032] The collagen used to create the cardiovascular components, e.g.,heart valves, e.g., semilunar heart valves, may be enriched withsignaling molecules which play a role in vascular development. Productsof three classes of genes are implicated: hedgehog, homeobox andcytokine. They include but are not limited to the following proteins:sonic hedgehog; NK-2, XNKx-3.3 (tinman), hCsx and Gax homeobox geneproducts; TGFbeta, VEGF, FGF, IGF, PDGF, and BMP4 cytokine proteins.Differentiation induced by the use of combinations of the foregoingproteins is promoted by incubation of the cell laden scaffold in vitrounder tissue culture conditions.

[0033] The term “growth factors” is art recognized and is intended toinclude, but is not limited to, one or more of platelet derived growthfactors (PDGF), e.g., PDGF AA, PDGF BB; insulin-like growth factors(IGF), e.g., IGF-I, IGF-II; fibroblast growth factors (FGF), e.g.,acidic FGF, basic FGF, β-endothelial cell growth factor, FGF 4, FGF 5,FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors (TGF), e.g.,TGF-β1, TGF-β1.2, TGF-β2, TGF-β3, TGF-β5; vascular endothelial growthfactors (VEGF), e.g., VEGF, epidermal growth factors (EGF), e.g., EGF,amphiregulin, betacellulin, heparin binding EGF; interleukins, e.g.,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14; colony stimulating factors (CSF), e.g., CSF-G,CSF-GM, CSF-M, BMP cytokine proteins; nerve growth factor (NGF); stemcell factor; hepatocyte growth factor, and ciliary neurotrophic factor.The term encompasses presently unknown growth factors that may bediscovered in the future, since their characterization as a growthfactor will be readily determinable by persons skilled in the art.

[0034] The term “extracellular matrix proteins” is art recognized and isintended to include one or more of fibronectin, laminin, vitronectin,tenascin, entactin, thrombospondin, elastin, gelatin, collagens,fibrillin, merosin, anchorin, chondronectin, link protein, bonesialoprotein, epinectin, hyaluronectin, undulin, epiligrin, and kalinin.The term encompasses presently unknown extracellular matrix proteinsthat may be discovered in the future, since their characterization as anextracellular matrix protein will be readily determinable by personsskilled in the art.

[0035] The term “proteoglycan” is art recognized and is intended toinclude one or more of decorin and dermatan sulfate proteoglycans,keratin or keratan sulfate proteoglycans, aggrecan or chondroitinsulfate proteoglycans, heparan sulfate proteoglycans, biglycan,syndecan, perlecan, or serglycin. The term encompasses presently unknownproteoglycans that may be discovered in the future, since theircharacterization as a proteoglycan will be readily determinable bypersons skilled in the art.

[0036] The term “glycosaminoglycan” is art recognized and is intended toinclude one or more of heparan sulfate, chondroitin sulfate, dermatansulfate, keratan sulfate, hyaluronic acid. The term encompassespresently unknown glycosaminoglycans that may be discovered in thefuture, since their characterization as a glycosaminoglycan will bereadily determinable by persons skilled in the art.

[0037] The term “polysaccharide” is art recognized and is intended toinclude one or more of heparin, dextran sulfate, chitin, alginic acid,pectin, and xylan. The term encompasses presently unknownpolysaccharides that may be discovered in the future, since theircharacterization as a polysaccharide will be readily determinable bypersons skilled in the art.

[0038] Suitable living cells include, but are not limited to, cellsderived from the layers of tissue comprising the semilunar heart valve,e.g., spongiosa, fibrosa, and ventricularis cells, epithelial cells, andmesothelial cells, e.g., keratinocytes, adipocytes, hepatocytes,neurons, glial cells, astrocytes, podocytes, mammary epithelial cells,islet cells; endothelial cells, e.g., aortic, capillary and veinendothelial cells; and mesenchymal cells, e.g., dermal fibroblasts,mesothelial cells, stem cells, osteoblasts, smooth muscle cells,striated muscle cells, ligament fibroblasts, tendon fibroblasts,chondrocytes, and fibroblasts.

[0039] In one embodiment, when the biopolymer is collagen, the collagencan be treated with an enzyme, e.g., lysyl oxidase which primes thecollagen for crosslinking. Lysyl oxidase, which can be purified from avariety of sources including, for example, calf aorta, human placenta,chicken embryo epiphyseal cartilage, pig skin, (see Shackleton, D. R.and Hulmes, D. J. S. (1990) Biochem. J. 266:917-919), and severallocations in pig embryos, converts the 68 -amino group of lysine to analdehyde. This aldehyde is a reactive functional group whichspontaneously binds to other lysine ε-amino groups or other aldehydes onother collagen molecules to form irreversible covalent crosslinks. Theresult is that collagen becomes insoluble. Lysyl oxidase can be added tothe collagen solutions under conditions which allow for the aldehydeconversion of the lysines. The lysyl oxidase is then removed from thecollagen solution and the collagen is processed as described hereinduring which the spontaneous crosslinks form. For example, during theprocessing of the collagen cardiovascular component, e.g., during thepolymerization step, the crosslinks spontaneously form as theconcentration of collagen per unit volume increases. Thelysyl-oxidase-mediated crosslink is strong, irreversible and is alinkage naturally found in collagen. Collagen crosslinked in this manneris insoluble and susceptible only to specific enzymatic attack duringremodeling of tissues. Lysyl oxidase can also be used to crosslinkcollagen for use as matt and matt compositions as well as spun fibers,gels, etc.

[0040] In still another embodiment, the strength of the cardiovascularcomponent can be increased by standard collagen crosslinking methodsusing, e.g., ultraviolet, dehydrothermal, or chemical crosslinkers.Typical chemical crosslinkers include, for example, glutaraldehyde,formaldehyde, acrylamide, carbodiimides, such as those known in the art,e.g., 1-ethyl-3-(dimethyaminopropyl)carbodiimide, diones known to thoseskilled in the art, e.g., 2,5-hexanedione, diimidates, e.g.,dimethylsuberimidate, or bisacrylamides, e.g.,N,N′-methylenebisacrylamide.

[0041] In still yet another embodiment of the invention, thecardiovascular components, e.g., heart valves, e.g., semilunar heartvalves, comprise biopolymer fiber scaffold derived from an aorticporcine valve processed in the absence of a crosslinking agent, e.g.,glutaraldehyde or chemicals similar thereto. By eliminating the step oftreating an aortic porcine valve with glutaraldehyde or with chemicalssimilar to glutaraldehyde, the calcification or structural breakdown ofthe aortic porcine valve tissue is eliminated. Accordingly, bindingsites for host human cells and other cells are maintained with thepresent invention.

[0042] Moreover another embodiment of the invention, the cardiovascularcomponents, e.g., heart valve, e.g., semilunar heart valve, comprise abiodegradable polymer fiber scaffold, e.g., a biopolymer fiber scaffold,having a structure determined by a digital program. The work of A. A. H.J. Sauren (The Mechanical behavior of the Aortic Valve (PhD thesis)Eindhoven, The Netherlands: Eindhoven Technical University, 1981), whichis incorporated herein by reference, has shown in whole mounts ofleaflets that the supporting scaffold of the leaflet consists ofcollagen fibers, having fractile properties, which extend from onecommisure to the other providing support for the applied load of blood.Equations which describe the fiber system of the leaflet have beenderived by C. S. Perkin and D. M. McQueen (Mechanical equilibriumdetermines the fractile fiber architecture of aortic heart valveleaflets. Am. J. Physiol. 266, H319-H328, 1994) from their functionwhich is to support a uniform load when the aortic valve is closed. Whatthey found is a single parameter family of collagen fibers with fractileproperties which compare closely with the whole mount fiberpreparations. Their work serves as the basis for creating a digitalprogram which a textile machine, or a sewing machine, could use toreproduce an approximation of the fiber scaffold of the valve leaflet.

[0043] The present invention also features a novel biocompatible annularsewing ring for attachment of a heart valve to the aortic wall of ahost. The annular ring is comprised of a biopolymer, e.g., collagen. Ina preferred embodiment, the biopolymer, e.g., collagen, is a biopolymerfiber, e.g., collagen fiber.

[0044] Referring to the figures, the semilunar valve of the aortaconsists of three leaflets or cusps which resemble pockets attached tothe wall of the aorta along their inferior edge (FIG. 1) with thesuperior edge of each pocket being free. If the wall of the aorta isslit between two leaflets along a vertical axis and the aorta is opened,three leaflets resembling pockets, all in a row, are seen attached tothe flattened aortic wall or root. The aorta or the aortic root and itsattached leaflets can be made in a mold; for example, a mold consistingof three parts which fit together tightly to make a closed aortic wallto which the three pocket like structures are attached (FIG. 4).

[0045] The parts of the mold for replicating a semilunar heart valve canbe constructed from inert materials, e.g., stainless steel or stainlesssteel coated with Teflon®. An example of such a mold is shown in FIG. 4.Referring to FIG. 4, the mold includes three integral parts, which areshown in views A, B, and C. The three parts fit together between twolateral edges to form a box with a hollow interior to form the aorticroot and attach to three valve leaflets by interconnecting U-shapedchannels. The first part (A) is shaped as a rectangular box cover whichrepresents the back or outside wall of the aorta, e.g., the aortic root.The second part (B) also employs a rectangular shape and fits togetherwith the first part A to form the front or inside wall of the aorta. Thesecond part (B) itself includes a front and back side which representthe front or inside walls and the back or outside walls, respectively,of the valve leaflets or cusps which attach to the front or inside wallof the aorta (A). The front or inside walls of the valve leaflets arerepresented on the front side of the second part (B) as hollow pocketswhich include the free edge, the nodulus arantis, the commisures, andthe lower edge of each valve leaflet. The commisures and the lower edgeof each valve leaflet, which form the hollow pockets, communicate withthe aorta and are represented by hollow U-shaped channels whichpenetrate the wall of the second part (B and C). The third part includesa plate (C) which forms the outside face of each valve leaflet, i.e.,the face intimal with the aorta. The third part completes the hollowchannels which form the valve leaflets (B′a and B′b) and includes aU-shaped curve structure that attaches to the back of the second part.The bottom of the U-shaped curve of the third part (B′a) includes awider diameter than the other areas of the communicating hollows (B′, C)to form the base or floor of the bottom edge of each valve leaflet.

[0046] With an assembled mold, sheets of a biodegradable polymer fiberscaffold, e.g., a biopolymer fiber scaffold, e.g., a collagen fiberscaffold, e.g., a woven (braided, knitted) collagen fiber scaffold, arelaid into the respective hollows (B) of the mold so that they lie incontact with the intimal surface of the aortic root and with the outsidesurface of the valve pockets, i.e., the surface away from the aorticwall. The sheets of a biodegradable polymer fiber scaffold, e.g., abiopolymer fiber scaffold, e.g., a collagen fiber scaffold, e.g., awoven (braided, knitted) collagen fiber scaffold, extend into the Ushaped slit and are bent to overlap the displaced plate (C) so that theylie in contact with the intimal surface of the aortic wall. Fiberscaffolds containing more than one type of biodegradable polymers canalso be formed, for example, by combining a biodegradable polymer fiberscaffold with a collagen fiber scaffold in the hollows of the mold asdescribed above.

[0047] In a preferred embodiment, a crosslinked collagen fiber scaffoldis laid into the respective hollows of the mold. In another preferredembodiment, a biopolymer fiber scaffold comprised of processed pig heartvalves can be laid into the respective hollows of the mold. In yetanother preferred embodiment, sheets of woven collagen threadsconstructed from a textile machine can be laid into the respectivehollows of the mold. As described previously, from equations for thecollagen fiber support system for the valve leaflet or cusp, a digitalprogram can be written for a textile or sewing machine which can producea collagen fiber scaffold in the form of three cusps using thecrosslinked collagen fibers described immediately above. Even withoutsuch a program from information already available the general pattern ofthe fiber system can be reproduced. For example each cusp can have thegeometry of the expanded cusp shown in FIG. 3, but cusps would beconnected at the commisures.

[0048] The hollows of the mold are then filled with collagen, e.g.,fetal porcine, fibrillar collagen, e.g., liquid dense fibrillarcollagen. In a preferred embodiment, the third part of the mold (C, B′b)is removed and the fiber reinforced collagen which has formed lining theaortic wall and the valve leaflet is seeded with cells which wouldnormally populate the respective tissues of the semilunar valve, e.g.,cells derived from the fibrosa, spongiosa, and ventricularis tissues.The mold can then be introduced into a cell culturing system to allowattachment, growth, and differentiation of the seeded cells. In anotherpreferred embodiment, the valve is seeded with cells which normallypopulate the outer tissue covering the semilunar valve, e.g., allogeneicor autogenous endothelial or mesothelial cells, to provide an epithelialcovering to the valves and aorta. These endothelial or mesothelial cellsare used to populate all tissue surfaces except the back or outersurface of the aortic wall after the first cells seeded into the tissuehave remodeled the biopolymer structure.

[0049] As described supra, the collagen, e.g., fetal porcine collagen,e.g., liquid dense fetal porcine collagen, used to fill the componentsof the mold may be enriched with signaling molecules which play a rolein vascular development. Cardiovascular complexes of signaling moleculescan be derived from the extracellular matrix of young or very youngporcine fetuses containing the proteins listed below. Products of threeclasses of genes are implicated: hedgehog, homeobox and cytokine. Theyinclude but are not limited to the following proteins: sonic hedgehog;NK-2, XNKx-3.3 (tinman), hCsx and Gax homeobox gene products; TGFbeta,VEGF, FGF, IGF, PDGF, and BMP4 cytokine proteins. Differentiationinduced by the use of combinations of the foregoing proteins is promotedby incubation of the cell laden scaffold in vitro under tissue cultureconditions.

[0050] The tissue which has formed can be lifted out of the mold and thetwo lateral edges of the valve are sewn together with a biopolymerthread, e.g., collagen or synthetic thread, to form a tubular valve. Ina preferred embodiment, additional endothelial or mesothelial cells maybe seeded onto the structure to cover the intimal surface of the aortaand the surfaces of the valve leaflets. In another embodiment, thetubular valve can be incorporated into a pulsatile closed circulatoryloop containing a nutrient fluid having the visco-elastic properties ofblood. The valve can be oriented in the circulation so that duringdiastole it back flows to fill the pockets of the valve. Mechanicalconditioning of the valve can be carried out for a period of 2-4 weeks.It can remain in culture ready for delivery on demand.

[0051] As described supra, the heart valves, e.g., semilunar valves, ofanimals, e.g., the pig heart, have been used as substitutes fordefective human valves for many years. The usual procedure forprocessing the valves after removal from an animal consists ofcrosslinking them with chemicals, e.g., glutaraldehyde. Chemicals suchas glutaraldehyde destroy all biological information associated with thescaffold of the valve. Accordingly, chemicals such as glutaraldehydecause the tissue of the removed valve to calcify and/or breakdownstructurally, thus, eliminating binding sites for cells, e.g., humanhost cells, which might otherwise be expected to seed into the leafletsof the valves as well as into the cuffs of the valves. There is also theloss of binding sites that would otherwise permit endothelial ormesothelial cells to populate the surfaces of the valve structures. Toovercome these limitations the following method has been developed toprocess the valves after removal from the donor animal. This methodeliminates the chemical crosslinking step, e.g., crosslinking withglutaraldehyde.

[0052] In a preferred embodiment, the valve, e.g., the semilunar valve,is removed from the donor animal, e.g., a pig. Using a 10% solution ofNAOH, cellular components are stripped from the collagen fiber scaffoldof the valve leaflets and annulus (the aortic wall), thus, eliminatingviruses, and other microorganisms, cells and cell surface antigenicdeterminants retaining the fibrous scaffold. The processed valve canthen be laid into a valve mold, e.g., the semilunar mold describedsupra, and be formed into a heart valve for transplantation.

[0053] In a preferred embodiment, the processed donor valve is laid intothe respective hollows (B) of the mold so that they lie in contact withthe intimal surface of the aortic root and with the outside surface ofthe valve pockets, i.e., the surface away from the aortic wall. Thedonor valve, e.g., pig valve, e.g., the pig semilunar valve, extend intothe U shaped slit and are bent to overlap the displaced plate (C) sothat they lie in contact with the intimal surface of the aortic wall.The hollows of the mold are then filled with collagen, e.g., fetalporcine collagen, or fibrillar collagen e.g., liquid dense fibrillarcollagen. In a preferred embodiment, the contents of the mold are freezedried to form a structure, e.g., a biopolymer foam, e.g., a collagenfoam around the donor valve. Methods for freeze drying collagen andforming biopolymer foams and collagen foams of varying densities aretaught in U.S. Pat. Nos. 5,891,558 and 5,709,934, each entitled“Biopolymer Foams for Use in Tissue Repair and Reconstruction,” thecontents of which are incorporated herein by reference.

[0054] In a preferred embodiment, as described supra, the third part ofthe mold (C, B′b) is removed and the donor valve reinforced foam whichhas formed lining the aortic wall and the valve leaflet is seeded withcells which would normally populate the respective tissues of thesemilunar valve, e.g., cells derived from the fibrosa, spongiosa, andventricularis tissues. The mold can then be introduced into a cellculturing system to allow attachment, growth, and differentiation of theseeded cells. In another preferred embodiment, the valve is seeded withcells which normally populate the outer tissue covering the semilunarvalve, e.g., allogeneic or autogenous endothelial or mesothelial cells,to provide an epithelial covering to the valves and aorta. Theseendothelial or mesothelial cells are used to populate all tissuesurfaces except the back or outer surface of the aortic wall after thefirst cells seeded into the tissue have remodeled the biopolymerstructure.

[0055] As described supra, the collagen, e.g., fetal porcine collagen,or fibrillar collagen, e.g., liquid dense fibrillar collagen, used tofill the components of the mold may be enriched with signaling moleculeswhich play a role in vascular development. Products of three classes ofgenes are implicated: hedgehog, homeobox and cytokine. They include butare not limited to the following proteins: sonic hedgehog; NK-2,XNKx-3.3 (tinman), hCsx and Gax homeobox gene products; TGFbeta, VEGF,FGF, IGF, PDGF, and BMP4 cytokine proteins. Differentiation induced bythe use of combinations of the foregoing proteins is promoted byincubation of the cell laden scaffold in vitro under tissue cultureconditions.

[0056] The tissue which has formed can be lifted out of the mold and thetwo lateral edges of the valve are sewn together with a biopolymerthread, e.g., collagen or synthetic thread, to form a tubular valve.

[0057] In a preferred embodiment, the valve is rehydrated in tissueculture medium and seeded with fibroblasts internally and withendothelial cells on its surfaces to demonstrate cell attachment,absence of toxicity, cell proliferation and differentiation. Thetoxicity and low information content of glutaraldehyde treated tissueswhich prevent the repopulation of processed valves are no longer aproblem when valves are prepared by the methods of this disclosure.

[0058] In another embodiment, the tubular valve can be incorporated intoa pulsatile closed circulatory loop containing a nutrient fluid havingthe visco-elastic properties of blood. The valve can be oriented in thecirculation so that during diastole it back flows to fill the pockets ofthe valve. Mechanical conditioning of the valve can be carried out for aperiod of 2-4 weeks or longer. It can remain in culture ready fordelivery on demand.

[0059] The invention also features a biocompatible annular sewing ringwhich can be used for attachment of a valve, e.g., a heart valve, e.g.,a semilunar valve, to the aorta. In one embodiment, a biopolymer clothor biopolymer matt, e.g., a collagen cloth or collagen matt, isprepared. Biopolymer and collagen matts are described in copendingpatent application Ser. No. 09/042,549, entitled “Biopolymer Matt forUse in Tissue Repair and Reconstruction,” the contents of which areincorporated herein by reference. The biopolymer cloth or matt, e.g.,collagen cloth or matt, can be wrapped around and stitched to abiopolymer matt in a rope-like structure, e.g., a collagen matt ropeshaped as a circle and stitched to the rope. Various shaped biopolymermatts, e.g., various shaped collagen matts, are described in copendingpatent application Ser. No. 09/042,549, entitled “Biopolymer Matt forUse in Tissue Repair and Reconstruction,” the contents of which areincorporated herein by reference. These, biopolymer matts, e.g.,collagen matts, can be cast into various shapes, e.g., tubes or orbs,such as spheres, to produce membranous structures which can containmaterial or liquids for specialized functions. Examples of implants madefrom matt, matt composite, or matt compositions include, for example,vessels, ducts, ureters, bladders and bone implants. Biopolymer matts,e.g., collagen matts, can be cast as tubes or orbs, such as spheres, toproduce membranous structures which can contain material or liquids forspecialized functions. Examples of implants made from matt, mattcomposite, or matt compositions include, for example, vessels, ducts,ureters, bladders and bone implants from matt cylinders filled with bonereplacement material.

[0060] In a preferred embodiment, the biocompatible annulus, e.g., thecollagen annulus, can be seeded with cells and enriched with signalingmolecules which play a role in vascular development. Products of threeclasses of genes are implicated: hedgehog, homeobox and cytokine. Theyinclude but are not limited to the following proteins: sonic hedgehog;NK-2, XNKx-3.3 (tinman), hCsx and Gax homeobox gene products; TGFbeta,VEGF, FGF, IGF, PDGF, and BMP4 cytokine proteins. Differentiationinduced by the use of combinations of the foregoing proteins is promotedby incubation of the cell laden scaffold in vitro under tissue cultureconditions. In another preferred embodiment, additional endothelial ormesothelial cells may be seeded onto the structure to cover the outersurface of the biocompatible annulus.

[0061] The contents of all cited references including literaturereferences, issued patents, published patent applications, andco-pending patent applications cited throughout this applicationincluding the background are hereby expressly incorporated by referencein their entirety.

[0062] Equivalents

[0063] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

What is claimed is:
 1. A semilunar heart valve, comprising abiodegradable polymer fiber scaffold and collagen.
 2. The semilunarheart valve of claim 1, wherein the biodegradable polymer fiber scaffoldis a biopolymer fiber scaffold.
 3. The semilunar heart valve of claim 1,wherein the collagen is porcine fetal collagen.
 4. The semilunar heartvalve of claim 1, wherein the collagen is fibrillar collagen.
 5. Thesemilunar heart valve of claim 4, wherein the fibrillar collagen isliquid dense fibrillar collagen.
 6. The semilunar heart valve of claim2, wherein the biopolymer fiber scaffold is a collagen biopolymerscaffold.
 7. The semilunar heart valve of claim 6, wherein the collagenis selected from the group consisting or collagen type I, collagen typeII, collagen type III, collagen type IV, collagen type V, collagen typeVI, collagen type VII, collagen type VIII, collagen type IX, collagentype X, collagen type XI, collagen type XII, collagen type XIII,collagen type XIV, and collagen type XVII.
 8. The semilunar heart valveof claim 6, wherein the collagen biopolymer scaffold is crosslinked. 9.The semilunar heart valve of claim 1, wherein the biodegradable polymerfiber scaffold is derived from an aortic porcine valve processed withouta crosslinking agent.
 10. The semilunar heart valve of claim 1, furthercomprising signaling molecules.
 11. The semilunar heart valve of claim1, wherein the polymer scaffold has a structure determined by a digitalprogram.
 12. A method of making a semilunar heart valve, comprising thesteps of: (a) assembling a mold which replicates the structure of asemilunar heart valve having between two lateral edges a hollowrepresenting the aortic root and hollows representing a plurality ofleaflets with outer and inner surfaces, the inner surfaces of thehollows representing the plurality of leaflets connecting with thehollow representing the aortic root and forming the intimal surface ofthe hollow representing the aortic root; (b) covering the intimalsurface of the hollow representing the aortic root and the outsidesurface of the hollow representing the plurality of leaflets with abiodegradable polymer fiber scaffold; (c) filling the hollowrepresenting the aortic root and the hollows representing the pluralityof leaflets with collagen; and (d) freeze-drying the polymer fiberscaffold and the collagen forming a tissue with two lateral edges. 13.The method of making a semilunar heart valve of claim 12, wherein thebiodegradable fiber scaffold is a biopolymer fiber scaffold.
 14. Themethod of making a semilunar heart valve of claim 12, wherein thecollagen is porcine fetal collagen.
 15. The method of making a semilunarheart valve of claim 12, wherein the collagen is fibrillar collagen. 16.The method of making a semilunar heart valve of claim 12, wherein thecollagen is enriched with signaling molecules.
 17. The method of makinga semilunar heart valve of claim 16, wherein the signaling molecules areselected from the group consisting of sonic hedgehog; NK-2, XNKx-3.3(tinman), hCsx and Gax homeobox gene products; TGFbeta, VEGF, FGF, IGF,PDGF, and BMP4 cytokine proteins.
 18. The method of making a semilunarheart valve of claim 12, further comprising the steps of removing thetissue from the mold, and sewing together the two lateral edges of thetissue.
 19. The method of making a semilunar heart valve of claim 12,further comprising the steps of, seeding the tissue with cells whichnormally populate human semilunar valve tissue.
 20. The method of makinga semilunar heart valve of claim 19, wherein the cells are selected fromthe group consisting of fibrosa, spongiosa, and ventricularis cells. 21.The method of making a semilunar heart valve of claim 21, furthercomprising the step of culturing the cells.
 22. The method of making asemilunar heart valve of either claim 12 or 19, further comprising thestep of seeding the tissue with endothelial or mesothelial cells.
 23. Anannular sewing ring for attachment of a heart valve to the aortic wallof a host, comprising: a biopolymer cloth and a biopolymer rope shapedin a circle, wherein the biopolymer cloth is wrapped around and stitchedto the biopolymer rope.
 24. The annular sewing ring of claim 23, whereinthe biopolymer fiber cloth is collagen cloth and the biopolymer rope iscollagen rope.
 25. The annular sewing ring of claim 24, wherein thecollagen is selected from the group consisting of collagen type I,collagen type II, collagen type III, collagen type IV, collagen type V,collagen type VI, collagen type VII, collagen type VIII, collagen typeIX, collagen type X, collagen type XI, collagen type XII, collagen typeXIII, collagen type XIV, and collagen type XVII.
 26. The annular sewingring of claim 23, wherein the ring is seeded with cells which normallypopulate semilunar valve tissue.
 27. The annular sewing ring of claim23, wherein the ring is enriched with signaling molecules.
 28. Theannular sewing ring of claim 23, wherein the signaling molecules areselected from the group consisting of sonic hedgehog; NK-2, XNKx-3.3(tinman), hCsx and Gax homeobox gene products; TGFbeta, VEGF, FGF, IGF,PDGF, and BMP4 cytokine proteins.
 29. A semilunar heart valve madeaccording to the method comprising the steps of: (a) assembling a moldwhich replicates the structure of a semilunar heart valve having betweentwo lateral edges a hollow representing the aortic root and hollowsrepresenting a plurality of leaflets with outer and inner surfaces, theinner surfaces connecting with the hollow representing the aortic rootand forming the intimal surface of the hollow representing the aorticroot; (b) covering the intimal surface of the hollow representing theaortic root and the outside surface of the hollow representing theplurality of leaflets with a biopolymer fiber scaffold; (c) filling thehollow representing the aortic root and the hollows representing theplurality of leaflets with collagen; and (d) freeze-drying thebiopolymer fiber scaffold and the liquid dense fibrillar collagenforming a tissue with two lateral edges.